ABSTRACT (30 Lines) Frozen section diagnoses are typically requested intraoperatively to guide the surgery. While this methodology is still irreplaceable, it suffers from a series of limitations: tissue loss, freezing artifacts, suboptimal cutting of fatty specimens, inability to diagnose bony lesions, and prolonged turnaround time. The aim is to develop a multi-channel subcellular resolution photoacoustic microscopy (PAM) system capable of addressing all the aforementioned limitations and providing pathologists with diagnostic images comparable or identical to the state of the art, with magnification matching or exceeding that of standard transmission light microscopes, and requiring no additional training for pathologists to diagnose. To achieve our objective, we will pursue the following two specific aims: Specific Aim 1: Develop a multi-channel subcellular resolution PAM system for rapid analysis and diagnosis of tissue biopsies A. Develop a multi-channel subcellular resolution PAM system B. Develop a customized tissue biopsy holder for rapid tissue biopsy handling and imaging by PAM Specific Aim 2: Optimize and validate the multi-channel subcellular resolution PAM for translation into a robust, accurate, and efficient intraoperative device A. Optimize image acquisition in order to maximize discrimination of nuclear, cytoplasmic, and stromal properties with minimal tissue degradation B. Optimize post-acquisition algorithms to generate pseudocolored images resembling classical hematoxylin and eosin (H&E) sections with superimposed unique imaging modalities (edema and collagen) C. Validate the utilization of PAM generated images for diagnostic purposes by comparing with H&E sections, which are prepared by formalin-fixed paraffin-embedding (the current gold standard) D. Validate the utilization of PAM generated images for intraoperative diagnostic purposes by comparing with H&E sections, which are prepared by frozen section (the current intraoperative gold standard) Impact: The proposed technology will represent a major breakthrough by addressing all the limitations of current frozen section-based diagnoses and providing images with H&E equivalent qualities intraoperatively. It aims to introduce a rapid and tissue saving diagnostic method for surgical biopsies, positively impacting the healthcare system and patient care. Reducing the turnaround time on the frozen section interpretation will significantly reduce the time spent in the operating room, and therefore, decrease patient risks and costs. The ability to evaluate adequacy of small specimens with no loss in tissue will decrease the need for re-biopsy procedures. The virtually generated images can be interpreted remotely so that smaller hospitals can benefit from procedures currently available only to larger tertiary hospitals and university centers.